This invention was made with Government support under Grant Nos. CA 30647, CA 42396 and CA 12582 awarded by the National Cancer Institute.
1. Field of the Invention
The present invention relates generally to Epstein-Barr virus-transformed human B-lymphoblastoid cell lines. More particularly, the present invention relates to such cell lines which are capable of producing antibodies which can be used to directly treat tumors or which can be used to raise anti-id antibodies for use as surrogate antigens or diagnostic reagents.
2. Description of Related Art
The publications and other reference materials referred to herein to describe the background of the invention and to provide additional detail regarding its practice are hereby incorporated by reference. For convenience, the reference materials are numerically referenced and grouped in the appended bibliography. The contents of these publications and other reference materials are hereby incorporated by reference.
The possibility that the variable regions of immunoglobulins could act as external antigens was first recognized by Jerne in his idiotype network theory (1). According to this theory, recognition of idiotypes on the antigen-combining site, or on the framework of AB1, results in the production of anti-idiotypes (anti-ids or AB2) beta and alpha, respectively. Such "internal image" anti-idiotypes, by virtue of their complementarity with the original antigen binding site, mimic the original antigen and often behave in a similar biological manner. The concept of internal image refers to the fact that some AB2 molecules can act as surrogate antigens and their administration can lead to the production of anti-anti-idiotype antibodies displaying similar characteristics of AB1.
Immunization using anti-ids as surrogate antigens has generated much interest among researchers, many of whom have experimented with AB2 vaccines for active specific immunization against viruses, bacteria, and other pathogens (2,3). This approach is useful when a conventional vaccine or antibodies are not available, or are difficult to produce or when the corresponding antigen is not a suitable product for genetic engineering. In addition, anti-ids can be used as immunomodulators for up-regulating immunity against cancer, and as immunosuppressants to prevent rejection of transplanted organs and to prevent the progression of auto-immune disease.
Gangliosides are glycospingolipids that are fundamental membrane components on human tissues. Gangliosides undergo characteristic changes during malignant transformation of normal cells and thus are desirable target antigens for immunotherapy of cancer. Melanoma synthesizes a large number of gangliosides and thus has served as a useful model to assess the potential of gangliosides as immunotherapy targets. A number of tumor-associated gangliosides of human melanoma and their respective immonogenicity have been defined (12-29). In addition, a number of studies have shown that active immunization with ganglioside antigens results in prolonged survival of melanoma patients (4,5). Nevertheless, this technique suffers in many areas, namely that the ganglioside antigen are many times rare or in short supply.
Tumor-associated antigens, in most cases, are present in nature only at low levels and are relatively difficult to purify in large amounts. In contrast, anti-ids can be secreted from hybridoma cells at low cost over long periods of time. Furthermore, current genetic engineering technology, while not applicable to ganglioside epitopes, can be used to synthesize the anti-id peptides. Anti-ids previously developed for active specific immunotherapy of human cancer have used murine monoclonal antibodies (MuMabs) as the immunogens (6-11).
In addition to their use as surrogate antigens, murine monoclonal antibodies have also been employed to define and characterize many antigenic molecules on human cancer cells. Murine monoclonal antibodies have several advantages over human monoclonal antibodies including a strong affinity for tumor antigens, higher antibody secretion by hybridoma ascites, and high antigen density on tumor cells. However, with respect to therapeutic use, recent clinical trials with murine monoclonals have indicated that human monoclonal antibodies (HuMAbs) may be preferable since repeated injections of MuMAbs induce anti-murine Ig antibodies in virtually all patients. This leads to formation of immune complexes and immune reactions with potentially hazardous complications. In addition, HuMAbs may recognize epitopes that are overlooked by the murine immune system.
The development of HuMAbs that react with ganglioside antigens on human cancer cells and the demonstration of their anti-tumor effect at the clinical level has been reported (23,12). Patients with recurrent melanoma received intratumor injections of HuMAb to ganglioside GD2 or GM2, and partial or complete regression was observed in about 70% of the patients. In those melanoma patients in whom the immunotherapy was ineffective, the target antigen GD2 or GM2, was not expressed on the tumor cells. Two HuMAbs identified as L55 and L72 have been produced from human B-lymphoblastoid cell lines which have been transformed by Epstein-Barr virus (29). The L55 and L72 antibodies were both found to be reactive with a variety of tumor cells.
Because the quantity and quality of gangliosides on human melanoma are widely heterogeneous between different cancer patients, it is desirable to avoid unnecessary administration of HuMAb by examination of a pre-treatment biopsy to identify which gangliosides dominate on each patient's tumor cells.
There are three different immunological assays which have been used to detect the quality and quantity of gangliosides present on a given tumor. They include: the immune adherence assay (IA); direct immunofluorescence with fluorescinated microspheres; and IA absorption, and a biochemical assay. These assays each have certain limitations and advantages. The immunologic assay requires single cell suspensions from the biopsied tumor tissues. However, it is often difficult to obtain viable high yield tumor cell populations. Also, under a light microscope, tumor cells may not be readily distinguished from monocytes and macrophages. The biochemical assay does not require intact cells. However, a relatively large volume of tumor is necessary for ganglioside extraction and measurement of sialic acid in the glycolipid preparation.
The most commonly utilized immunologic technique for defining antigen expression on biopsy specimens using murine monoclonal antibodies is immunohistochemical staining of tissue sections. However, this sensitive method is not readily applicable to combinations of human monoclonal antibodies and human tissues. The indirect staining of human tumor tissues with the second antibody (anti-human Ig) usually results in high background from non-specific binding to abundant endogenous human Ig. Direct immunostaining using biotinylated human monoclonal antibodies may overcome this high background (30). However, this method is usually less sensitive and is most effective when a high density antigen is present on the cell surface.
There presently is a continuing need to develop additional human cell lines which are capable of producing antibody that is immunogenic with respect to the gangliosides present on tumors. The anti-ganglioside antibodies produced by the new cell lines will be useful in direct treatment of tumors and also in the production of anti-ids for use as surrogate antigens or diagnostic reagents.